Reactive oxygen species (ROS) are produced by normal physiological processes in the body and play a significant role in the flow energy and information in all the living systems. (Voeikov (2001) Riv. Biol. 94:237-258). ROS also perform critical functions related to intercellular induction of apoptosis (Bauer (2000) Anticancer Res. 20:4115-4139), inflammation processes and immune responses. ROS are also generated as by-products of normal metabolic processes, from food additives, from environmental sources, such as ultraviolet radiation (Wenk et al. (2001) Curr. Probl. Dermatol. 29:83-94) and tobacco smoke (Stich et al. (1991) American Journal of Clinical Nutrition 53:298S-304S), and from many other pollutants. Reactive oxygen species (ROS) include oxygen related free radicals, such as superoxide (O2−.), peroxyl (ROO−.), alkoxyl (RO−.), hydroxyl (HO−.), and nitric oxide (NO−.); and non-radical species, such as the singlet oxygen (1O2), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl).
Cells have specific mechanisms to maintain homeostasis, which keep ROS levels in check. (Mates (2000) Toxicology 153:83-104). A number of different biological pathways are employed to maintain oxidative homeostasis within a cell. These pathways include the synthesis and recycling of γ-glutamyl-cysteinyl-glycine (Glutathion GSH) and the action of specific enzymes, such as SOD, catalases and peroxidases. (Deneke et al. (1989) Am. J. Physiol. 257: L163-L173). The generation and recycling of GSH is commonly known as the γ-glutamyl cycle. (Lieberman et al. (1995) Amer. J. Pathol. 147:1175-1185). This cycle, which is illustrated in FIG. 1, culminates in the production of the naturally occurring intracellular anti-oxidant, GSH. The maintenance of appropriate levels of GSH is very important to the redox state of the cell.
Superoxide anions are among the most reactive and damaging ROS produced by the mitochondria. Consequently, the regulation of their production and neutralization is a very important component of maintaining the cellular redox state. The enzyme superoxide dismutase (SOD) catalyzes the production of the less reactive hydrogen peroxide from superoxide anions, as illustrated by equation 1. The hydrogen peroxide produced is subsequently reduced to water by either catalase or glutathione peroxidase (GPx). (Wei et al. (2001) Chin. J. Physiol. 31:1-11).                               2          ⁢                      O                          2              ·                        -                          ⁢                              →                          2              ⁢                                                           ⁢                              H                +                                              SOD                ⁢                                            H              2                        ⁢                          O              2                                +                      O            2                                              (        1        )            
If the processes that maintain oxidative homeostasis in the cell get out of balance, free radical levels become dangerous, as these are highly reactive, molecules that damage DNA, proteins and components on cell membranes, eventually leading to cellular damage throughout the body and contributing a primary role in the aging process.
As a result of our modern diet and lifestyle, exposure to free radicals is increasing dramatically having a profound effect on our vulnerability to disease. The role of oxidative stress and its associated age related diseases is well established. Physiological changes that occur as we age, result in the loss of a homeostatic balance between the generation of ROS, which cause oxidative damage and the production of naturally occurring antioxidants, such as glutathione (GSH) and other regulatory enzymes (superoxide dismutase, catalase and peroxidases). As the origin of these ROS is the mitochondria, this loss of homeostasis that occurs during aging is known as the “mitochondrial theory of aging” (Simon (2000) Annals New York Acad. Sci. 908:219-225). Characteristic changes within the mitochondria during aging include, a decrease in the expression of the enzymes Cu/Zn-superoxide dismutase (SOD), which are responsible for “neutralizing” highly reactive and oxidative superoxide anions, accumulation of hydrogen peroxide and the reduction of mitochondrial glutathione pools. There is also a loss of intracellular and plasma GSH levels, resulting in an increasingly global oxidative environment within the human body during aging. This environment translates to a number of problems on both the cellular level and the level of the organism itself, as evidenced by the generation of chronic diseases of aging associated with oxidative damage.
For example, decreased GSH levels have been found in people suffering from debilitating neuro-degenerative diseases such as Alzheimer's Disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Cellular changes within individual cells include, physical damage to cellular components, as well as, alterations in cellular responses resulting from the increasingly oxidative environment. The physical damage includes, the oxidative damage to both cellular and mitochondrial DNA and the peroxidation of lipids within the cellular and mitochondrial membranes. These changes affect the integrity of both of these components and translate to impaired function. The oxidative environment also contributes to changes in many gene responses. (Forsberg et al. (2001) Arch. Biochem. Biophys. 389: 84-93). This is because transcription factors, such as NF-kB and AP-1 are controlled by changes in the redox state of cells. For example, NF-kB transcription factors are activated in oxidative environments. Thus, minute alterations in the redox state that occur during aging could dramatically change the way a cell responds to a particular stimulus. The altered responses can manifest themselves as increased apoptosis, cancers or the loss of function that ultimately cause many of the diseases related to aging. (Zs.-Nagy (2001) Annals New York Acad. Sci. 928:187-199).
ROS also a play critical role after brain injury, implicating them in the pathology of traumatic CNS damage and cerebral ischemia. (Lewen (2000), J. Neurotrauma 17:871-890). ROS caused oxidative stress in endothelial cells is a leading condition in the pathogenesis of many cardiovascular diseases (Touyz (2000) Curr. Hypertens. Rep. 2:98-105), pulmonary diseases (Berry et al. (2001) Curr. Opin. Nephrol. Hypertens. 10:247-255) and metabolic disorders (Takahashi (2000) Nippon Rinsho 58:1592-1597).
Protection of alveolar epithelial cells and vascular endothelia cells against pulmonary (Muzykantov (2001) Antioxid. Redox Signal 3: 39-62) and vascular endothelium oxidative stress (Muzykantov (2001) J. Control Release 12:1-21) has been investigated via delivery antioxidative enzymes, such as SOD and catalase. The beneficial effects from dietary antioxidants, such as resveratrols (Hung et al. (2002) Br. J. Pharmacol. 135:1627-1633) and alpha-lipoic acid (Takaoka et al. (2002) Clin. Exp. Pharmacol. 29:189-194) in reducing the incidence of coronary heart diseases, butylated hydroxytoluene and β-carotene in photocarcinogenesis (Black (2002) Front Biosci. 7:D1044-1055) has been documented. Even though antioxidants may reduce free radicals generated by radiotherapy and chemotherapy, there is no evidence suggesting that they interfere with conventional cancer therapy. Clinical evidence indicates that cancer patients given antioxidants exhibit higher tolerance and decreased side effects, resulting from treatment and further that they live longer and have a higher quality of life. (Lamson et al. (1999) Altern. Med. Rev. 4:304-329). While these diseases represent extreme examples, it is well documented that the skewing of the redox state toward an oxidative environment is a characteristic of the aging process. Therefore, the average person benefits by maintaining a homeostatic oxidative balance. Because the oxidative damage that occurs during the aging process can be directly linked to pathological aspects of these diseases, controlling or restoring the homeostatic balance of the oxidative state is of great interest to the medical industry as a whole. Consequently, anti-oxidants have a secure place within the Dietary Supplemental Health and Education Act (DSHEA) arena, as evidenced by the number of antioxidant products targeted for the anti-aging market.
Antioxidant defense mechanisms are species specific and heavily influenced by nutrition, since important antioxidants, such as, ascorbic acid and α-tocopherol cannot be synthesized by humans and therefore, must be obtained from ones diet. (Benzie (2000) Eur. J. Nutr. 39:53-61). Antioxidants are very popular dietary supplements in the nutritional and cosmeceutical industries. Types of products promoted as antioxidants include, vitamins (i.e., Vc, Ve, Vb, β-carotene), minerals (i.e., selenium), amino acids (i.e., lysine, cysteine, n-acetyl cysteine, lipoic acid), phenolic acids (i.e., curcumin, rosveratrol, chetechins, EGCG), flavanoids (i.e., rutin, quercetine, etc.) (Pietta (2000) J. Nat. Prod. 63:1035-1042), anthrocynadines, pycnogenol, coumarine derivatives, polyphenols (i.e., tannins) and many different types of botanical extracts. Antioxidant products include polyunsaturated fatty acids (PUFAs) and specialty amino acids, claims for which range from reducing the risk of heart disease to treating joint problems and easing depression. β-carotene is a major antioxidant, which has been shown to reduce the risk of prostate cancer. β-carotene is primarily available in dietary supplements, specifically multivitamin formulations and single-entity soft gel capsules. The product is a popular antioxidant, thought to help prevent many diseases.
Lycopene, an up-and-coming phytochemical in the β-carotene family, is receiving increased attention because of its promise as an antioxidant. The new product, Lycopene 5% TG, is readily available in multivitamin, antioxidant, straight and chewable formulations. Sabinsa Corp. has developed a new, colorless tumeric root extract, tetrahydrocurcuminoids (THC), for use in dietary supplements and cosmeceuticals as a bioprotectant and multipurpose antioxidant that does not stain. THC is a free radical scavenger, preventing free radical chain reactions by neutralizing existing free radicals and/or maintaining a reducing environment around the cells and preventing the formation of free radicals; and acting as a chelating agent to generate non-active complexes with prooxidatant metals. It is claimed that some antioxidants enhance the protective capability of the cell wall, thereby bolstering the cell's defense against free radicals and repairing damage done to cells by free radicals. However, there is not much scientifically sound data to prove the efficacy of these antioxidants.
The Oxygen Radical Absorption Capacity (ORAC) assay is a method for measuring total serum antioxidant activity. (Cao et al. (1993) Free Rad. Biol. Med. 14:303-311). It can be used to quantitatively measure the total antioxidant capacity, as well as, qualitatively measure the levels of fast versus slow acting antioxidants in a blood serum sample. The assay utilizes the free radical sensitive fluorescent indicator protein β-phycoerythrin (β-PE) to monitor the effectiveness of various serum antioxidants in protecting β-PE from becoming damaged by free radicals. Assay results are quantitated by allowing the reaction to reach completion and then integrating the area under the kinetic curve relative to a blank reaction containing no antioxidant. The area under the curve is proportional to the concentration of all the antioxidants present in the sample. (DeLang and Glazer (1989) Analyt. Biochem. 177:300-306).
The Lipid Peroxidation Inhibition Capacity (LPIC) assay is a method for measuring the ability of a sample to inhibit the initiation and propagation of a spontaneous lipid peroxidation reaction. Lipid peroxidation represents the major mechanism of lipid destruction occurring in an organism. This reaction is also an important source of reactive oxygen species. In part, lipid peroxidation is controlled in vivo by:
1) the chelation of trace metals, such as iron and copper, that are involved in the initiation of lipid peroxidation reactions; and
2) the presence of antioxidants that terminate the propagation reaction once it is initiated.
Lipid peroxidation is believed to be one of the major destructive reactions, occurring in the plasma components and blood vessel walls, leading the onset of cardiovascular disease. (Riemersma et al. (1991) Lancet 337:1-5). Thus, a principal role of antioxidants and metal-chelating components in the serum is the protection of the entire cardiovascular system through the control of lipid peroxidation. (Stampfer et al. (1993) New England Journal of Medicine 328:1444-1449; Rimm et al. (1993) New England Journal of Medicine 328:1450-1456). Metal chelators, such as ferritin for iron and metallothionein for copper are well known, but other serum constituents, such as urate, which can chelate iron, may also be important. (Maples et al. (1988) J. Biol. Med. 263:1709-1712). Antioxidants, such as α-tocopherol, are known to prevent propagation of lipid peroxidation reactions, but there may be many other serum constituents that could be equally or even more important than α-tocopherol. (Stahelin et al. (1984) Journal of the National Cancer Institute 73:1463-1468). The LPIC assay measures how well a sample is able to both inhibit and terminate a lipid peroxidation reaction. The LPIC value complements the ORAC value. The ORAC assay provides information on total antioxidant capacity, whereas the LPIC assay provides information on both total antioxidant capacity and on the status of metal catalysts in effectively protecting against in vivo lipid peroxidation reactions.
Chromones are a specific type of aromatic compounds having a benzopyran-4-one as their major skeletal structure as illustrated by the following general structure: wherein
R1, R2 and R3 are independently selected from the group consisting of —H, —OH, —CH3, —SH, alkyl, alkenyl, oxoalkyl, oxoalkenyl, hydroxylalkyl, hydroxylalkenyl, —OCH3, —SCH3, —OR, —SR, —NH2, —NRH, —NR2, —NR3+X−, esters of the mentioned substitution groups, including, but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; thereof carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, but not limited to, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized chromones;
wherein said alkyl and/or alkenyl group is a straight and/or branched chain having between 1-20 carbon atoms with and/or without double bonds in different positions;
X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.; and
R is an alkyl group having between 1-20 carbon atoms.
To date there are only 183 chromones isolated from natural sources. (The Combined Chemical Dictionary, Chapman & Hall/CRC, Version 5:1 June 2001).
Chromones reportedly exhibit monoamine oxidase inhibitory activity (Fujimoto et al. (2002) Chem. Pharm. Bull. 50:330-336), tyrosinase inhibitory activity (Piao et al. (2002) Chem. Pharm. Bull. 50:309-311), anti-platelet effects (Leoncini et al. (1991) Pharmacol. Res. 23:139-148), inhibitory activity of phosphatidylinositol-3-kinase (Pong et al. (1998) J. Neurochem. 71:1912-1919; Blommaart et al. (1997) Eur. J. Biochem 243:240-246), growth inhibitory activity against oral pathogens (Cai (1996) J. Nat. Prod. 59:987-990), prostaglandin H synthase inhibitory activity (Jurenka et al. (1989) Comp. Biochem. 93:253-255). Chromones also possess therapeutic efficacy against type II collagen-induced arthritis in rats (Inaba et al. (2000) Chem. Pharm. Bull. 48:131-139) and hypolipidemic activity (Witiak et al. (1975) J. Med. Chem. 18:934-942; Tetko et al. (1995) Bioorg Khim. 21:809-815). It has also been reported that chromones can function as selective sigma receptor ligands (Erickson et al. (1992) J. Med. Chem. 35:1526-1535). Based on animal studies, chromones are easily absorbed and metabolized (Crew et al. (1976) Xenobiotica 6:89-100) and the c-glucosyl bond of aloesin can be cleaved by human intestinal bacteria. (Che et al. (1991) Chem. Pharm. Bull. 39:704-708).
Aloe is an intricate plant that contains many biologically active substances. (Cohen et al. in Wound Healing/Biochemical and Clinical Aspects, 1st ed. W B Saunders, Philadelphia (1992)). Over 300 species of Aloe are known, most of which are indigenous to Africa. Studies have shown that the biologically active substances are located in three separate sections of the aloe leaf—a clear gel fillet located in the center of the leaf, in the leaf rind or cortex of the leaf and in a yellow fluid contained in the pericyclic cells of the vascular bundles, located between the leaf rind and the internal gel fillet, referred to as the latex. Historically, Aloe products have been used in dermatological applications for the treatment of burns, sores and other wounds. These uses have stimulated a great deal of research in identifying compounds from Aloe plants that have clinical activity, especially anti-inflammatory activity. (See, e.g., Grindlay and Reynolds (1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. of Ethnopharmacology 23:61-71). As a result of these studies there have been numerous reports of Aloe compounds having diverse biological activities, including anti-tumor activity, anti-gastric ulcer, anti-diabetic, anti-tyrosinase activity (see, e.g., Hirata and Suga et al. (1977) Z. Naturforsch 32c:731-734) and antioxidant activity (Yu and Lee, U.S. Pat. No. 5,939,395).
Chromones isolated from various Aloe species have been reported to have diverse biological activity. Aloesin (FIG. 2) reportedly inhibits tyrosinase activity (Jones et al. Journal of Pigment Cell Research, Acceptance, Feb. 10th. 2002) and up-regulates cyclin E-dependent kinase activity (Lee et al. (1997) Biochem. Mol. Biol. Int. 41:285-292). A c-glycosyl chromone isolated from Aloe barbadensis demonstrates anti-inflammatory activity (Hutter et al. (1996) J. Nat. Prod. 59:541-543) and antioxidant activity similar to that of alpha-tocopherol based on a rat brain homogenates model (Lee et al. Free Radic Biol. Med. 28:261-265).
Aloe barbadensis leaves and its bitter principles exhibit effects on blood glucose level in normal and alloxan diabetic mice (Ajabnoor (1990) J. Ethnopharmacol. 28:215-220) and dried sap of various Aloe species demonstrates anti-diabetic activity in clinical studies (Ghannam, (1986) Horm Res. 24:288-294).
Yagi et al. disclose a group of compounds isolated from Aloe, particularly aloesin and one of its derivatives, 2″-O-feruloylaloesin, which are effective inhibitors of tyrosinase. (Yagi et al. (1987) Plant Medica 515-517). Biochemical testing of the enzyme inhibition by means of the Lineweaver Burk diagram showed that 2″-feruloylaloesin was a non-competitive inhibitor of tyrosinase, while aloesin is a competitive inhibitor. Aloesin is a C-glucosylated 5-methylchromone (Holdsworth (1972) Chromones in Aloe Species, Part I-Aloesin PM 19(4):322-325). In vitro, aloesin is a strong inhibitor of tyrosinase activity (Yagi et al. (1987) Planta Medica 515-517). In assays of tyrosinase activity on the substrate L-DOPA, aloesin is capable of 50% inhibition at a concentration of 0.2 mM.
U.S. Pat. No. 6,083,976, entitled “Method of Synthesis of Derivatives of Aloesin,” describes a novel method for the synthesis of derivatives of aloesin alkylated at the C-7 hydroxyl group. The alkylated aloesins, produced by this method have the functionality of aloesin, a tyrosinase-inhibiting compound with skin whitening activity, but have greater biological activity than aloesin as indicated by in vitro tyrosinase assays. Additionally, the alkyl group makes the derivatized aloesins more fat soluble than aloesin, allowing them to be retained in the stratum corneum of the skin more effectively than aloesin. As a result, the alkylated aloesins are more potent and faster acting skin lightening agents than aloesin.
U.S. Pat. No. 6,123,959, entitled “Aqueous Composition Comprising Active Ingredients for the De-Pigmentation of the Skin,” describes aqueous compositions comprising liposomes of phospholipids, and at least one competitive inhibitor of an enzyme for the synthesis of melanin, in combination with at least one non-competitive inhibitor of an enzyme for the synthesis of melanin. The competitive inhibitors of the invention include aloesin and derivatives thereof. The invention also includes the use of the compositions for the de-pigmentation of skin. Each of these patents is incorporated herein by reference in their entirety.
To date, known methods for purifying aloesin, as well as, other chromones involve the use of chromatography. (See e.g., Rauwald and Beil (1993) J. of Chromatography 639:359-362; Rauwald and Beil (1993) Z. Naturforsch 48c:1-4; Conner et al. (1990) Phytochemistry 29:941; Holdsworth (1972) Chromones in Aloe Species, Part I-Aloesin PM 19(4):322-325; Mebe (1987) Phytochemistry 26:2646; Haynes et al. (1970) J. Chem. Soc. (C) 2581; McCarthy and Haynes (1967) The Distribution of Aloesin in Some South African Aloe Species; Heft 3 342). These procedures were developed for chemical analysis and are not practical for preparative scale production of aloesin. In U.S. patent application Ser. No. 09/792,104, filed Feb. 26, 2001, entitled “Method of Purification of Aloesin,” which is incorporated herein by reference in its entirety, a method for purification of aloesin using crystallization is disclosed. Applicant knows of no report or suggestion of a method for the purification of chromones from Aloe and other indicated species using with polyamide or LH-20 column chromatography.